Background-Circulating levels of interleukin-6 (IL-6) and tumor necrosis factor-␣ (TNF-␣) are elevated in diabetic patients. We assessed the role of glucose in the regulation of circulating levels of IL-6, TNF-␣, and interleukin-18 (IL-18) in subjects with normal or impaired glucose tolerance (IGT), as well as the effect of the antioxidant glutathione. Methods and Results-Plasma glucose levels were acutely raised in 20 control and 15 IGT subjects and maintained at 15 mmol/L for 5 hours while endogenous insulin secretion was blocked with octreotide. In control subjects, plasma IL-6, TNF-␣, and IL-18 levels rose (PϽ0.01) within 2 hours of the clamp and returned to basal values at 3 hours. In another study, the same subjects received 3 consecutive pulses of intravenous glucose (0.33 g/kg) separated by a 2-hour interval. Plasma cytokine levels obtained at 3, 4, and 5 hours were higher (PϽ0.05) than the corresponding values obtained during the clamp. The IGT subjects had fasting plasma IL-6 and TNF-␣ levels higher (PϽ0.05) than those of control subjects. The increase in plasma cytokine levels during the clamping lasted longer (4 hours versus 2 hours, PϽ0.01) in the IGT subjects than in the control subjects, and the cytokine peaks of IGT subjects after the first glucose pulse were higher (PϽ0.05) than those of control subjects. On another occasion, 10 control and 8 IGT subjects received the same glucose pulses as above during an infusion of glutathione; plasma cytokine levels did not show any significant change from baseline after the 3 glucose pulses. Conclusions-Hyperglycemia acutely increases circulating cytokine concentrations by an oxidative mechanism, and this effect is more pronounced in subjects with IGT. This suggests a causal role for hyperglycemia in the immune activation of diabetes.

The effects of intermittent and constant high glucose in the formation of nitrotyrosine and 8-hydroxydeoxyguanosine (markers of oxidative stress), as well as the possible linkage between oxidative stress and apoptosis in endothelial cells, have been evaluated. Stable high glucose increased nitrotyrosine, 8-hydroxydeoxyguanosine (8-OHdG), and apoptosis levels. However, these effects were more pronounced in intermittent high glucose. Protein kinase C (PKC) was elevated in both such conditions, particularly in intermittent glucose. The adding of the PKC inhibitors bisindolylmaleimide-I and LY379196, a specific inhibitor of PKC-␤ isoforms, normalized nitrotyrosine and reduced 8-OHdG concentration and cell apoptosis in both stable and intermittent high glucose. Similar results were obtained with the MnSOD mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride that normalized nitrotyrosine, 8-OHdG, and apoptosis and inhibited PKC activation. NAD(P)H oxidase was also measured. NAD(P)H oxidase components p47phox, p67phox, and p22phox was overexpressed during both stable and intermittent high glucose. PKC inhibition and MnSOD mimetic normalized this phenomenon. In conclusion, our study shows that the exposure of endothelial cells to both stable and intermittent high glucose stimulates reactive oxygen species overproduction also through PKC-dependent activation of NAD(P)H oxidase, leading to increased cellular apoptosis. Our data suggest that glucose fluctuations may also be involved in the development of vascular injury in diabetes. Diabetes 52:2795-2804, 2003

Background-Postprandial hypertriglyceridemia and hyperglycemia are considered risk factors for cardiovascular disease.Evidence suggests that postprandial hypertriglyceridemia and hyperglycemia induce endothelial dysfunction through oxidative stress; however, the distinct role of these two factors is a matter of debate. Methods and Results-Thirty type 2 diabetic patients and 20 normal subjects ate 3 different meals: a high-fat meal; 75 g glucose alone; and high-fat meal plus glucose. Glycemia, triglyceridemia, nitrotyrosine, and endothelial function were assayed during the tests. Subsequently, diabetics took 40 mg/d simvastatin or placebo for 12 weeks. The 3 tests were performed again at baseline, between 3 to 6 days after the start, and at the end of each study. High-fat load and glucose alone produced a decrease of endothelial function and an increase of nitrotyrosine in normal and diabetic subjects. These effects were more pronounced when high fat and glucose were combined. Short-term simvastatin treatment had no effect on lipid parameters but reduced the effect on endothelial function and nitrotyrosine observed during each different test. Long-term simvastatin treatment was accompanied by a lower increase in postprandial triglycerides, which was followed by smaller variations of endothelial function and nitrotyrosine during the tests. Conclusions-This study shows an independent and cumulative effect of postprandial hypertriglyceridemia and hyperglycemia on endothelial function, suggesting oxidative stress as common mediator of such effect. Simvastatin shows a beneficial effect on oxidative stress and endothelial dysfunction, which may be ascribed to a direct effect as well as the lipid-lowering action of the drug.

To explore the effect of fluctuating glucose on endothelial cells, human umbilical vein endothelial cells were incubated for 14 days in media containing different glucose concentrations: 5 mmol/l, 20 mmol/l, or a daily alternating 5 or 20 mmol/l glucose. Apoptosis was studied by different methods: viability assay, cell cycle analysis, DNA fragmentation, and morphological analysis. Furthermore, the levels of Bcl-2 and Bax, well known proteins involved in apoptosis, were evaluated. Stable high glucose induced apoptosis in human umbilical vein endothelial cells, a phenomenon accompanied by a significant decrease of Bcl-2 and a simultaneous increase of Bax expression. However, apoptosis was enhanced in human umbilical vein endothelial cells exposed to intermittent, rather than constant, high glucose concentration. In this condition, Bcl-2 was not detectable, whereas Bax expression was significantly enhanced. These findings suggest that variability in glycemic control could be more deleterious to endothelial cells than a constant high concentration of glucose.

Adhesion molecules, particularly intracellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin, have been associated with cardiovascular disease. Elevated levels of these molecules have been reported in diabetic patients. Postprandial hypertriglyceridemia and hyperglycemia are considered risk factors for cardiovascular disease, and evidence suggests that postprandial hypertriglyceridemia and hyperglycemia may induce an increase in circulating adhesion molecules. However, the distinct role of these two factors is a matter of debate. Thirty type 2 diabetic patients and 20 normal subjects ate three different meals: a high-fat meal, 75 g of glucose alone, and a high-fat meal plus glucose. Glycemia, triglyceridemia, plasma nitrotyrosine, ICAM-1, VCAM-1, and Eselectin were assayed during the tests. Subsequently, diabetic subjects took simvastatin 40 mg/day or placebo for 12 weeks. The three tests were performed again at baseline, between 3 and 6 days after starting the study, and at the end of each study. High-fat load and glucose alone produced an increase of nitrotyrosine, ICAM-1, VCAM-1, and E-selectin plasma levels in normal and diabetic subjects. These effects were more pronounced when high fat and glucose were combined. Short-term simvastatin treatment had no effect on lipid parameters, but reduced the effect on adhesion molecules and nitrotyrosine, which was observed during every different test. Long-term simvastatin treatment was accompanied by a lower increase in postprandial triglycerides, which was followed by smaller variations in ICAM-1, VCAM-1, E-selectin, and nitrotyrosine during the tests. This study shows an independent and cumulative effect of postprandial hypertriglyceridemia and hyperglycemia on ICAM-1, VCAM-1, and E-selectin plasma levels, suggesting oxidative stress as a common mediator of such effects. Simvastatin shows a beneficial effect on oxidative stress and the plasma levels of adhesion molecules, which may be ascribed to a direct effect in addition to the lipid-lowering action of the drug. Diabetes 53: [701][702][703][704][705][706][707][708][709][710] 2004

Background-Postprandial hypertriglyceridemia and hyperglycemia are considered risk factors for cardiovascular disease.Evidence suggests that postprandial hypertriglyceridemia and hyperglycemia induce endothelial dysfunction and inflammation through oxidative stress. Statins and angiotensin type 1 receptor blockers have been shown to reduce oxidative stress and inflammation, improving endothelial function. Methods and Results-Twenty type 2 diabetic patients ate 3 different test meals: a high-fat meal, 75 g glucose alone, and a high-fat meal plus glucose. Glycemia, triglyceridemia, endothelial function, nitrotyrosine, C-reactive protein, intercellular adhesion molecule-1, and interleukin-6 were assayed during the tests. Subsequently, diabetics took atorvastatin 40 mg/d, irbesartan 300 mg/d, both, or placebo for 1 week. The 3 tests were performed again between 5 and 7 days after the start of each treatment. High-fat load and glucose alone produced a decrease in endothelial function and increases in nitrotyrosine, C-reactive protein, intercellular adhesion molecule-1, and interleukin-6. These effects were more pronounced when high-fat load and glucose were combined. Short-term atorvastatin and irbesartan treatments significantly counterbalanced these phenomena, and their combination was more effective than either therapy alone. Conclusions-This study confirms an independent and cumulative effect of postprandial hypertriglyceridemia and hyperglycemia on endothelial function and inflammation, suggesting oxidative stress as a common mediator of such an effect. Short-term treatment with atorvastatin and irbesartan may counterbalance this phenomenon; the combination of the 2 compounds is most effective.

Background It has been previously shown that hyperglycemia enhances free radical production, inducing oxidative damage, which in its turn activates the death pathways implicated in cell apoptosis and necrosis. But the possible involvement of this pathway in the hyperglycemia-induced apoptosis of endothelial cells has not yet been reported.

This study investigated coronary perfusion pressure, nitric oxide (NO) and superoxide production, nitrotyrosine (NT) formation, and cardiac cell apoptosis in isolated hearts perfused with high glucose concentration. Coronary perfusion pressure; NO and superoxide anion generation; immunostaining for NT, inducible NO synthase (iNOS), and the constitutive type of NO synthase (NOS) eNOS; iNOS and eNOS mRNA expression by Western blot and RT-PCR; and apoptosis of cardiac cells were studied in hearts perfused for 2 h with solutions containing D-glucose at a concentration of 11.1 mmol/l (control), D-glucose at the concentration of 33.3 mmol/l (high glucose), or D-glucose (33.3 mmol/l) plus glutathione (0.3 mmol/l). Perfusion of isolated hearts in conditions of high glucose concentration caused a significant increase of coronary perfusion pressure (P < 0.001) and an increase of both NO and superoxide generation. However, superoxide production was 300% higher than baseline, whereas NO production was 40% higher (P < 0.001 for both). This effect was accompanied by the formation of NT, and an increase of iNOS expression. eNOS remained unchanged. At the end of the experiments, cardiac cell apoptosis was evident in hearts perfused with high glucose. The effects of high glucose were significantly prevented by glutathione. This study demonstrates that high glucose for 2 h is enough to increase iNOS gene expression and NO release in working rat hearts. Upregulation of iNOS and raised NO generation are accompanied by a marked concomitant increase of superoxide production, a condition favoring the production of peroxynitrite, a powerful pro-oxidant that can mediate the toxic effects of high glucose on heart by itself and/or via the formation of nitrotyrosine, as suggested by the detection of cell apoptosis.